The long-term goal of this research is to understand how bacterial autolysis contributes to biofilm formation. Bacteria are commonly found in the environment as complex biofilm communities. This has clinical significance as pathogens often form biofilms during infection and protect the pathogen from the immune system and antibiotics. Recently, controlled cell death and lysis modulated by the cidABC and IrgAB operons have been shown to be important in Staphylococcus aureus biofilm formation. In the current application, two of the proteins encoded in these operons, CidB and LrgB, will be examined. These proteins are wide-spread, being found in 46 percent of sequenced genomes including bacteria and archaea, yet none have a predicted or assigned function. The specific aims of this application are to: 1) characterize mutants in the genes encoding these proteins using assays of cell survival and autolysis, antibiotic tolerance, and biofilm production;and, 2) examine the cellular localization and protein-protein interactions that may be important for function. The results of the experiments outlined in this study will examine the function of two previously uncharacterized proteins believed to be involved in controlling cell death and lysis. This will provide insight into the poorly understood process of programmed cell death in bacteria and how this process contributes to the establishment of a biofilm. Finally, it may suggest new avenues to pursue for the development of novel antibacterial agents. PUBLIC HEALTH RELEVANCE: Bacterial infections are a growing threat to human health around the world primarily due to their increasing ability to cause infections recalcitrant to antibiotic therapy. The research described in this application focuses on the novel concept that bacteria regulate their own cell death as part of complex developmental processes. The results generated by these studies will provide a better understanding of the molecular mechanisms controlling bacterial cell death and will lead to novel and more effective therapeutic strategies to combat infections.